Maraviroc, celastrol and azelastine alter Chlamydia trachomatis development in HeLa cells.

Introduction . Chlamydia trachomatis (Ct) is an obligate intracellular bacterium, causing a range of diseases in humans. Interactions between chlamydiae and antibiotics have been extensively studied in the past.Hypothesis/Gap statement: Chlamydial interactions with non-antibiotic drugs have received less attention and warrant further investigations. We hypothesized that selected cytokine inhibitors would alter Ct growth characteristics in HeLa cells.Aim. To investigate potential interactions between selected cytokine inhibitors and Ct development in vitro.Methodology. The CCR5 receptor antagonist maraviroc (Mara; clinically used as HIV treatment), the triterpenoid celastrol (Cel; used in traditional Chinese medicine) and the histamine H1 receptor antagonist azelastine (Az; clinically used to treat allergic rhinitis and conjunctivitis) were used in a genital in vitro model of Ct serovar E infecting human adenocarcinoma cells (HeLa).Results. Initial analyses revealed no cytotoxicity of Mara up to 20 µM, Cel up to 1 µM and Az up to 20 µM. Mara exposure (1, 5, 10 and 20 µM) elicited a reduction of chlamydial inclusion numbers, while 10 µM reduced chlamydial infectivity. Cel 1 µM, as well as 10 and 20 µM Az, reduced chlamydial inclusion size, number and infectivity. Morphological immunofluorescence and ultrastructural analysis indicated that exposure to 20 µM Az disrupted chlamydial inclusion structure. Immunofluorescence evaluation of Cel-incubated inclusions showed reduced inclusion sizes whilst Mara incubation had no effect on inclusion morphology. Recovery assays demonstrated incomplete recovery of chlamydial infectivity and formation of structures resembling typical chlamydial inclusions upon Az removal.Conclusion. These observations indicate that distinct mechanisms might be involved in potential interactions of the drugs evaluated herein and highlight the need for continued investigation of the interaction of commonly used drugs with Chlamydia and its host.

Water Filtered Infrared A and Visible Light (wIRA/VIS) Irradiation Reduces Chlamydia trachomatis Infectivity Independent of Targeted Cytokine Inhibition.

Chlamydia trachomatis is the major cause of infectious blindness and represents the most common bacterial sexually transmitted infection worldwide. Considering the potential side effects of antibiotic therapy and increasing threat of antibiotic resistance, alternative therapeutic strategies are needed. Previous studies showed that water filtered infrared A alone (wIRA) or in combination with visible light (wIRA/VIS) reduced C. trachomatis infectivity. Furthermore, wIRA/VIS irradiation led to secretion of pro-inflammatory cytokines similar to that observed upon C. trachomatis infection. We confirmed the results of previous studies, namely that cytokine secretion (IL-6, IL-8, and RANTES/CCL5) upon wIRA/VIS treatment, and the subsequent reduction of chlamydial infectivity, are independent of the addition of cycloheximide, a host protein synthesis inhibitor. Reproducible cytokine release upon irradiation indicated that cytokines might be involved in the anti-chlamydial mechanism of wIRA/VIS. This hypothesis was tested by inhibiting IL-6, IL-8, and RANTES secretion in C. trachomatis or mock-infected cells by gene silencing or pharmaceutical inhibition. Celastrol, a substance derived from Trypterygium wilfordii, used in traditional Chinese medicine and known for anti-cancer and anti-inflammatory effects, was used for IL-6 and IL-8 inhibition, while Maraviroc, a competitive CCR5 antagonist and anti-HIV drug, served as a RANTES/CCL5 inhibitor. HeLa cell cytotoxicity and impact on chlamydial morphology, size and inclusion number was evaluated upon increasing inhibitor concentration, and concentrations of 0.1 and 1 µM Celastrol and 10 and 20 µM Maraviroc were subsequently selected for irradiation experiments. Celastrol at any concentration reduced chlamydial infectivity, an effect only observed for 20 µM Maraviroc. Triple dose irradiation (24, 36, 40 hpi) significantly reduced chlamydial infectivity regardless of IL-6, IL-8, or RANTES/CCL5 gene silencing, Celastrol or Maraviroc treatment. Neither gene silencing nor pharmaceutical cytokine inhibition provoked the chlamydial stress response. The anti-chlamydial effect of wIRA/VIS is independent of cytokine inhibition under all conditions evaluated. Thus, factors other than host cell cytokines must be involved in the working mechanism of wIRA/VIS. This study gives a first insight into the working mechanism of wIRA/VIS in relation to an integral component of the host immune system and supports the potential of wIRA/VIS as a promising new tool for treatment in trachoma.

The Chlamydia suis Genome Exhibits High Levels of Diversity, Plasticity, and Mobile Antibiotic Resistance: Comparative Genomics of a Recent Livestock Cohort Shows Influence of Treatment Regimes.

Chlamydia suis is an endemic pig pathogen, belonging to a fascinating genus of obligate intracellular pathogens. Of particular interest, this is the only chlamydial species to have naturally acquired genes encoding for tetracycline resistance. To date, the distribution and mobility of the Tet-island are not well understood. Our study focused on whole genome sequencing of 29 C. suis isolates from a recent porcine cohort within Switzerland, combined with data from USA tetracycline-resistant isolates. Our findings show that the genome of C. suis is very plastic, with unprecedented diversity, highly affected by recombination and plasmid exchange. A large diversity of isolates circulates within Europe, even within individual Swiss farms, suggesting that C. suis originated around Europe. New World isolates have more restricted diversity and appear to derive from European isolates, indicating that historical strain transfers to the United States have occurred. The architecture of the Tet-island is variable, but the tetA(C) gene is always intact, and recombination has been a major factor in its transmission within C. suis. Selective pressure from tetracycline use within pigs leads to a higher number of Tet-island carrying isolates, which appear to be lost in the absence of such pressure, whereas the loss or gain of the Tet-island from individual strains is not observed. The Tet-island appears to be a recent import into the genome of C. suis, with a possible American origin.

Water-filtered infrared A reduces chlamydial infectivity in vitro without causing ex vivo eye damage in pig and mouse models.

Repeated ocular infections with Chlamydia trachomatis trigger the development of trachoma, the most common cause of infectious blindness worldwide. Water-filtered infrared A (wIRA) has shown positive effects on cultured cells and human skin. Our aim was to evaluate the potential of wIRA as a possible non-chemical treatment for trachoma patients. We both modeled ocular chlamydial infections using C. trachomatis B to infect human conjunctival epithelial cells (HCjE) and studied the effects of wIRA on non-infected ocular structures with two ex vivo eye models. We focused on the temperature development during wIRA irradiation in cell culture and perfused pig eyes to exclude potentially harmful side effects. Furthermore, cell viability of HCjE and cytotoxicity in mouse retina explants was analyzed. We demonstrated a significant wIRA-dependent reduction of chlamydial infectivity in HCjE cells. Moreover, we observed that wIRA treatment of HCjE prior to infection was sufficient to inhibit chlamydial infectivity and that visible light enhances the effect of wIRA. Irradiation did not reduce cell viability and there was no indication of retinal damage post treatment. Additionally, temperatures during wIRA exposure did not markedly exceed physiological eye temperatures, suggesting that hyperthermia-related lesions are unlikely. For clinical applications, further exploration of wIRA as a non-chemical treatment device in an experimental animal model is essential.

Water-filtered infrared a irradiation in combination with visible light inhibits acute chlamydial infection.

New therapeutic strategies are needed to overcome drawbacks in treatment of infections with intracellular bacteria. Chlamydiaceae are Gram-negative bacteria implicated in acute and chronic diseases such as abortion in animals and trachoma in humans. Water-filtered infrared A (wIRA) is short wavelength infrared radiation with a spectrum ranging from 780 to 1400 nm. In clinical settings, wIRA alone and in combination with visible light (VIS) has proven its efficacy in acute and chronic wound healing processes. This is the first study to demonstrate that wIRA irradiation combined with VIS (wIRA/VIS) diminishes recovery of infectious elementary bodies (EBs) of both intra- and extracellular Chlamydia (C.) in two different cell lines (Vero, HeLa) regardless of the chlamydial strain (C. pecorum, C. trachomatis serovar E) as shown by indirect immunofluorescence and titration by subpassage. Moreover, a single exposure to wIRA/VIS at 40 hours post infection (hpi) led to a significant reduction of C. pecorum inclusion frequency in Vero cells and C. trachomatis in HeLa cells, respectively. A triple dose of irradiation (24, 36, 40 hpi) during the course of C. trachomatis infection further reduced chlamydial inclusion frequency in HeLa cells without inducing the chlamydial persistence/stress response, as ascertained by electron microscopy. Irradiation of host cells (HeLa, Vero) neither affected cell viability nor induced any molecular markers of cytotoxicity as investigated by Alamar blue assay and Western blot analysis. Chlamydial infection, irradiation, and the combination of both showed a similar release pattern of a subset of pro-inflammatory cytokines (MIF/GIF, Serpin E1, RANTES, IL-6, IL-8) and chemokines (IL-16, IP-10, ENA-78, MIG, MIP-1α/ß) from host cells. Initial investigation into the mechanism indicated possible thermal effects on Chlamydia due to irradiation. In summary, we demonstrate a non-chemical reduction of chlamydial infection using the combination of water-filtered infrared A and visible light.